Polypeptides having organophosphorous hydrolase activity

ABSTRACT

The present invention relates to isolated polypeptides having organophosphorous hydrolase activity, and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND

1. Field of the Invention

The present invention relates to polypeptides having organophosphoroushydrolase activity, and polynucleotides encoding the polypeptides. Theinvention also relates to nucleic acid constructs, vectors, and hostcells comprising the polynucleotides as well as methods of producing andusing the polypeptides.

2. Description of the Related Art

Organophosphorous compounds are known in the art. In particular somewarfare agents are known to be organophosphorous compounds such as theG-type nerve agents such as Sarin, Cyclosarin, and Soman and the V-typenerve agents such as VX. Other organophosphorous compounds are known aspesticides.

It is desirable to be able to decontaminate areas contaminated with suchorganophosphorous compounds. A polypeptide having organophosphoroushydrolase activity, such as diisopropylfluorophosphatase activity hasbeen suggested for this purpose since such polypeptides are capable ofhydrolyzing harmful organophosphorous compounds and thereby convertingthem to less harmful products.

In WO 99/43791, a diisopropylfluorophosphatase from Loligo vulgaris isdisclosed and its potential use for decontamination among otherapplications is also described.

WO 2009/130285, WO 2010/128115 and WO 2010/128116 disclose otherdiisopropylfluorophosphatases from Pseudoalteramonas haloplanktis,Octopus vulgaris, and Aplysia califomica.

It is an object of the present invention to provide polypeptides havingorganophosphorous hydrolase e.g. diisopropylfluorophosphatase activityand polynucleotides encoding the polypeptides, in particular having highstability and/or high specific activity.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingorganophosphorous hydrolase activity selected from the group consistingof:

-   (a) a polypeptide having at least 65% sequence identity to the    mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or    SEQ ID NO: 8;-   (b) a polypeptide encoded by a polynucleotide that hybridizes under    medium stringency conditions with (i) the mature polypeptide coding    sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO:    7, or (ii) the full-length complement of (i);-   (c) a polypeptide encoded by a polynucleotide having at least 65%    sequence identity to the mature polypeptide coding sequence of SEQ    ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7;-   (d) a variant of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO:    4, SEQ ID NO: 6, or SEQ ID NO: 8, comprising a substitution,    deletion, and/or insertion at one or more (e.g., several) positions;    and-   (e) a fragment of the polypeptide of (a), (b), (c), or (d) that has    organophosphorous hydrolase activity.

The present invention also relates to isolated polynucleotides encodingthe polypeptides of the present invention; nucleic acid constructs;recombinant expression vectors; recombinant host cells comprising thepolynucleotides; and methods of producing the polypeptides.

The present invention also relates to methods for removing anorganophosphorous compound, comprising contacting the organophosphorouscompound with a polypeptide of the invention.

Definitions

Organophosphorous hydrolase: The term “organophosphorous hydrolase” isdefined herein as hydrolytic activity to organophosphorous compounds, inparticular phosphorous anhydride bonds in organophosphorous compoundsincluding nerve gases. Thus the term includes an enzyme with hydrolaseactivity and/or esterase activity, e.g. organophosphorous hydrolaseactivity (such as an organophosphoesterase activity) or organophosphoricacid anhydrolase (OPAA) activity, or carboxylesterase activity,diisopropylfluorophosphatase (DFPase) activity (EC 3.1.8.2),dehalogenase activity, catalyzing the hydrolyses of phosphorus-sulfurbonds, prolidase activity and/or imidodipeptidase activity.

The term “DFPase (EC3.1.8.2)” is defined herein asdiisopropylfluorophosphatase, dialkylfluorophosphatase,diisopropylphosphorofluoridate hydrolase, diisopropylfluorophosphonatedehalogenase, diisopropylphosphofluoridase,isopropylphosphorofluoridase, organophosphate acid anhydrase,organophosphorous acid anhydrolase, somanase, tabunase. DFPases acts onphosphorus anhydride bonds (such as phosphorus-halide andphosphorus-cyanide) in organophosphorous compounds (including nervegases).

For purposes of the present invention, organophosphorous hydrolaseactivity is determined according to the procedure described in Example3. In one aspect, the polypeptides of the present invention have atleast 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%,at least 80%, at least 90%, at least 95%, or at least 100% of theorganophosphorous hydrolase activity of the mature polypeptide of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

Decontamination: The term “decontamination” is to be understood hereinas removing harmful agents such as organophosphorous compounds, e.g.nerve gases, toxins, pesticides, thus the term includes e.g.detoxification activity.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Fragment: The term “fragment” means a polypeptide or a catalytic domainhaving one or more (e.g., several) amino acids absent from the aminoand/or carboxyl terminus of a mature polypeptide or domain; wherein thefragment has organophosphorous hydrolase activity. In one aspect, afragment contains at least 302 amino acid residues (e.g., amino acids 12to 314 of SEQ ID NO: 2, or amino acids 7 to 309 of SEQ ID NO: 4).

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5× SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. It is known in the art that a hostcell may produce a mixture of two of more different mature polypeptides(i.e., with a different C-terminal and/or N-terminal amino acid)expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving organophosphorous hydrolase activity.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5× SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5× SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and either 35%formamide, following standard Southern blotting procedures for 12 to 24hours. The carrier material is finally washed three times each for 15minutes using 2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the—nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the—nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having organophosphorous hydrolase activity. In one aspect, asubsequence contains at least 906 nucleotides (e.g., nucleotides 34 to942 of SEQ ID NO: 1, or nucleotides 19 to 927 of SEQ ID NO: 3).

Variant: The term “variant” means a polypeptide having organophosphoroushydrolase activity comprising an alteration, i.e., a substitution,insertion, and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5× SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

DETAILED DESCRIPTION

Polypeptides having Organophosphorous Hydrolase Activity

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8 of at least 65%, e.g., atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which haveorganophosphorous hydrolase activity. In one aspect, the polypeptidesdiffer by no more than 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or9, from the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, or SEQ ID NO: 8.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,or SEQ ID NO: 8; or SEQ ID NO: 9 to SEQ ID NO: 26; or SEQ ID NO: 27 toSEQ ID NO: 31; or an allelic variant thereof; or is a fragment thereofhaving organophosphorous hydrolase activity. In another aspect, thepolypeptide comprises or consists of the mature polypeptide of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; or SEQ ID NO: 9 toSEQ ID NO: 26; or SEQ ID NO: 27 to SEQ ID NO: 31.

In another embodiment, the present invention relates to an isolatedpolypeptide having organophosphorous hydrolase activity encoded by apolynucleotide that hybridizes under medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7,or (ii) the full-length complement of (i) (Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.).

The polynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQID NO: 7, or a subsequence thereof, as well as the polypeptide of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, or a fragmentthereof, may be used to design nucleic acid probes to identify and cloneDNA encoding polypeptides having organophosphorous hydrolase activityfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having organophosphorous hydrolase activity.Genomic or other DNA from such other strains may be separated by agaroseor polyacrylamide gel electrophoresis, or other separation techniques.DNA from the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that hybridizes with SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, or a subsequence thereof, thecarrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7; (ii)the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, or SEQ ID NO: 7; (iii) the full-length complement thereof;or (iv) a subsequence thereof; under medium to very high stringencyconditions. Molecules to which the nucleic acid probe hybridizes underthese conditions can be detected using, for example, X-ray film or anyother detection means known in the art.

In another embodiment, the present invention relates to an isolatedpolypeptide having organophosphorous hydrolase activity encoded by apolynucleotide having a sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ IDNO: 7 of at least 65%, e.g., at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQID NO: 8, comprising a substitution, deletion, and/or insertion at oneor more (e.g., several) positions. In an embodiment, the number of aminoacid substitutions, deletions and/or insertions introduced into themature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQID NO: 8 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9.Preferably, the substitution, deletion, and/or insertion are onlysubstitutions. The amino acid changes may be of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of 1-30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to 20-25 residues; or a smallextension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for organophosphorous hydrolase activity toidentify amino acid residues that are critical to the activity of themolecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708.The active site of the enzyme or other biological interaction can alsobe determined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity ofessential amino acids can also be inferred from an alignment with arelated polypeptide.

In an embodiment, essential amino acids in the amino acid sequence ofSEQ ID NO: 2 are located at positions E25, N132, N190, and D239. Otheressential positions in SEQ ID NO: 2 may be N133, T204, S282, N283, andH298.

In another embodiment, essential amino acids in the amino acid sequenceof SEQ ID NO: 4 are located at positions E20, N127, N185, and D234.Other essential positions in SEQ ID NO: 4 may be N128, T199, S277, N278,and H293.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld, 4: 35-48.

Sources of Polypeptides having Organophosphorous Hydrolase Activity

A polypeptide having organophosphorous hydrolase activity of the presentinvention may be obtained from microorganisms of any genus. For purposesof the present invention, the term “obtained from” as used herein inconnection with a given source shall mean that the polypeptide encodedby a polynucleotide is produced by the source or by a strain in whichthe polynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

The polypeptide may be a Phlebobranchia (a suborder of sea squirts inthe order Enterogona) polypeptide. Preferably, it is a Cionidaepolypeptide; for example, the polypeptide may be a Ciona polypeptidesuch as a Ciona edwardsi, Ciona fascicularis, Ciona gelatinosa, Cionaimperfect, Ciona intestinalis, Ciona mollis, or Ciona savignyipolypeptide.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from the above sources,or DNA samples obtained using the above-mentioned probes. Techniques forisolating DNA directly from natural habitats are well known in the art.A polynucleotide encoding the polypeptide may then be obtained bysimilarly screening a genomic DNA or cDNA library. Once a polynucleotideencoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques thatare known to those of ordinary skill in the art (see, e.g., Sambrook etal., 1989, supra).

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention, as described herein.

The techniques used to isolate or clone a polynucleotide are known inthe art and include isolation from genomic DNA or cDNA, or a combinationthereof. The cloning of the polynucleotides from genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligation activated transcription (LAT) andpolynucleotide-based amplification (NASBA) may be used. Thepolynucleotides may be cloned from a strain of Ciona, or a relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the polynucleotide.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for synthesizing polypeptides substantiallysimilar to the polypeptide. The term “substantially similar” to thepolypeptide refers to non-naturally occurring forms of the polypeptide.These polypeptides may differ in some engineered way from thepolypeptide isolated from its native source, e.g., variants that differin specific activity, thermostability, pH optimum, or the like. Thevariants may be constructed on the basis of the polynucleotide presentedas the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, or SEQ ID NO: 7, e.g., a subsequence thereof, and/or byintroduction of nucleotide substitutions that do not result in a changein the amino acid sequence of the polypeptide, but which correspond tothe codon usage of the host organism intended for production of theenzyme, or by introduction of nucleotide substitutions that may giverise to a different amino acid sequence. For a general description ofnucleotide substitution, see, e.g., Ford et al., 1991, ProteinExpression and Purification 2: 95-107.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide priorto its insertion into a vector may be desirable or necessary dependingon the expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMR1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In a preferred aspect, the cell is a Ciona cell. In a more preferredaspect, the cell is a Ciona savignyi or Ciona intestinalis cell.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cell may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). If the polypeptide is secreted into thenutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it can be recovered fromcell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideof the present invention so as to express and produce a polypeptide ordomain in recoverable quantities. The polypeptide or domain may berecovered from the plant or plant part. Alternatively, the plant orplant part containing the polypeptide or domain may be used as such forimproving the quality of a food or feed, e.g., improving nutritionalvalue, palatability, and rheological properties, or to destroy anantinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing the polypeptide or domainmay be constructed in accordance with methods known in the art. Inshort, the plant or plant cell is constructed by incorporating one ormore expression constructs encoding the polypeptide or domain into theplant host genome or chloroplast genome and propagating the resultingmodified plant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide or domain operablylinked with appropriate regulatory sequences required for expression ofthe polynucleotide in the plant or plant part of choice. Furthermore,the expression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide or domainis desired to be expressed. For instance, the expression of the geneencoding a polypeptide or domain may be constitutive or inducible, ormay be developmental, stage or tissue specific, and the gene product maybe targeted to a specific tissue or plant part such as seeds or leaves.Regulatory sequences are, for example, described by Tague et al., 1988,Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide or domain in the plant. For instance, thepromoter enhancer element may be an intron that is placed between thepromoter and the polynucleotide encoding a polypeptide or domain. Forinstance, Xu et al., 1993, supra, disclose the use of the first intronof the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are hereinincorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a polypeptide or domain canbe introduced into a particular plant variety by crossing, without theneed for ever directly transforming a plant of that given variety.Therefore, the present invention encompasses not only a plant directlyregenerated from cells which have been transformed in accordance withthe present invention, but also the progeny of such plants. As usedherein, progeny may refer to the offspring of any generation of a parentplant prepared in accordance with the present invention. Such progenymay include a DNA construct prepared in accordance with the presentinvention. Crossing results in the introduction of a transgene into aplant line by cross pollinating a starting line with a donor plant line.Non-limiting examples of such steps are described in U.S. Pat. No.7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideor domain of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide or domain under conditions conducive for production ofthe polypeptide or domain; and (b) recovering the polypeptide or domain.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. The compositions may be preparedin accordance with methods known in the art and may be in the form of aliquid or a dry composition. For instance, the polypeptide compositionmay be in the form of a granulate or a microgranulate. The polypeptideto be included in the composition may be stabilized in accordance withmethods known in the art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

The present invention is also directed to methods for using thepolypeptides having organophosphorous hydrolase activity(organophosphorous hydrolases), or compositions thereof.

In one preferred embodiment the invention also directed to the use oforganophosphorous hydrolases of the invention for decontamining an areaor a device contaminated with at least one harmful or undesiredorganophosphorous compound. The organophosphorous hydrolases of theinvention or a composition comprising the organophosphorous hydrolasesof the invention is applied to the area or the device in an amountsufficient to degrade at least part of at least one harmful or undesiredorganophosphorous compound.

In another embodiment the organophosphorous hydrolases of the inventionmay be used emulsions such as micro emulsions for applying to e.g. ahuman or animals. The organophosphorous hydrolases of the invention or acomposition comprising the organophosphorous hydrolases of the inventionis applied to the human or animal to protect against at least oneharmful or undesired organophosphorous compound.

In a further embodiment the organophosphorous hydrolases of theinvention may be incorporated in an assay for detection of at least oneharmful or undesired organophosphorous compound. Such assays could bebeneficial for quick assessment of the presence of undesiredorganophosphorous compound

Harmful or undesired organophosphorous compounds includes toxicorganophosphorous cholinesterase-inhibiting compounds including nervegases (G agents or G-series) such as ethylN,N-dimethylphosphoramidocyanidate (tabun), diisopropylfluorophosphate(DFP), O-isopropyl methylphosphonofluoridate (sarin), O-pinacolyl methylphosphonofluoridate (soman) and O-cyclohexyl methylphosphonofluoridate.

Other harmful compounds includes V agents (or V-series), which maycomprise VX, VE, VG, VM, VR Tetriso and Soviet V-gas (Russian VX).

The pesticides may comprise fungicides, insecticides, herbicide androdenticides. The pesticide may be Demeton-S, Demeton-S-methyl,Demeton-S-methylsulphon, Demeton-methyl, Parathion, Phosmet,Carbophenothion, Benoxafos, Azinphos-methyl, Azinphos-ethyl, Amiton,Amidithion, Cyanthoate, Dialiphos, Dimethoate, Dioxathion, Disulfoton,Endothion, Etion, Ethoate-methyl, Formothion, Malathion, Mercarbam,Omethoate, Oxydeprofos, Oxydisulfoton, Phenkapton, Phorate, Phosalone,Prothidathion, Prothoate, Sophamide, Thiometon, Vamidothion,Methamidophos.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Example 1 Cloning and Expression of Organophosphorous Hydrolase GeneCloning

Synthetic genes encoding the His-tagged organophosphorous hydrolasesfrom Ciona savigny (CSAV) (SEQ ID NO: 6) and Ciona intestinalis (CINT)(SEQ ID NO: 8) were designed and the genes were synthesized by acommercial supplier. Subsequently an expression construct for expressionof the organophosphorous hydrolases was created using standard methodsof molecular biology (Sambrook et al. (1989); Ausubel et al. (1995);Harwood and Cutting (1990) and this was integrated by homologousrecombination into a Bacillus subtilis host cell genome. The geneconstruct was expressed under the control of a triple promoter system(as described in WO 99/43835), consisting of the promoters from Bacilluslicheniformis alpha-amylase gene (amyL), Bacillus amyloliquefaciensalpha-amylase gene (amyQ), and the Bacillus thuringiensis cryIIIApromoter including the mRNA stabilizing sequence. The gene coding forChloramphenicol acetyl-transferase was used as marker.

Cloning of Variants of Ciona savigny DFPase

17 variants containing the following single amino acid changes in theCiona savigny DFPase (SEQ ID NO: 6) were cloned and expressed: M63A (SEQID NO: 9), M63G (SEQ ID NO: 10), R107I (SEQ ID NO: 11), R107V (SEQ IDNO: 12), R107L (SEQ ID NO: 13), A109S (SEQ ID NO: 14), A109C (SEQ ID NO:15), E178F (SEQ ID NO: 16), E178I (SEQ ID NO: 17), E178L (SEQ ID NO:18), E178V (SEQ ID NO: 19), R180F (SEQ ID NO: 20), R180I (SEQ ID NO:21), R180L (SEQ ID NO: 22), R180V (SEQ ID NO: 23), R180M (SEQ ID NO:24), Y276H (SEQ ID NO: 25), and Y276F (SEQ ID NO: 26).

Cloning of Variants of Ciona intestinalis DFPase

5 variants containing the following single amino acid changes in theCiona intestinalis DFPase (SEQ ID NO: 8) were cloned and expressed:E173F (SEQ ID NO: 27), E173V (SEQ ID NO: 28), R175A (SEQ ID NO: 29),S60L (SEQ ID NO: 30), and S60R (SEQ ID NO: 31).

Cloning of Variants of CSAV and CINT DFPases

To generate variants of SEQ ID NO: 6 and SEQ ID NO: 8, PCR-basedsite-directed mutagenesis was done with a mutagenic primer (see Table 1)that introduce the desired sequence change (substitutions). Primers weredesigned so that the mutation lies in the middle of the oligonucleotidewith sufficient flanking nucleotides (15-25). The Bacillus genomic DNAwith CSAV DFPase and CINT DFPase was used as template and PCR was setupwith a proofreading DNA polymerase (Phusion DNA polymerase (New EnglandBiolabs). Subsequently, an expression construct for expression of theorganophosphorous hydrolases were created using standard methods ofmolecular biolgy (Sambrook et al. (1989); Ausubel et al. (1995); Harwoodand Cutting (1990) and this was integrated by homologous recombinationinto a Bacillus subtilis host cell genome. The gene construct wasexpressed under the control of a triple promoter system (as described inWO 99/43835), consisting of the promoters from Bacillus licheniformisalpha-amylase gene (amyL), Bacillus amyloliquefaciens alpha-amylase gene(amyQ), and the Bacillus thuringiensis cryIIIA promoter including themRNA stabilizing sequence. The gene coding for Chloramphenicolacetyl-transferase was used as marker. Correct sequences were verifiedby sequencing colony PCR products from Bacillus subtilis transformants.

TABLE 1 Mutagenic primers used for PCR-based site-directedmutagenesis of Ciona savigny DFPase. Bold lettersrepresent sites of directed mutagenesis. SEQ ID Mutation NO.Mutagenic primer M63A 325′-CGGTTCTCGTCCGCAGCCTCAGCAGGCGCAACCGCATAGAAGCG-3′ M63G 335′-CGGTTCTCGTCCGCAGCCTCACCAGGCGCAACCGCATAGAAGCG-3′ R107I 345′-GGTCTGATTGACAACCAGCAGGGATACCTCCATAGCCGTCAAAATGAG-3′ R107V 355′-GGTCTGATTGACAACCAGCAGGAACACCTCCATAGCCGTCAAAATGAG-3′ R107L 365′-GGTCTGATTGACAACCAGCAGGAAGACCTCCATAGCCGTCAAAATGAG-3′ A109S 375′-CCTCATGGTCTGATTGACAACCTGAAGGGCGACCTCCATAGCCGTC-3′ A109C 385′-CCTCATGGTCTGATTGACAACCACAAGGGCGACCTCCATAGCCGTC-3′ E178F 395′-CAGTAAAGATAGTCTCGCGGTCAAAAGGTGAAGGTGCGATAGGTGATC-3′ E178I 405′-CAGTAAAGATAGTCTCGCGGTCGATAGGTGAAGGTGCGATAGGTGATC-3′ E178L 415′-CAGTAAAGATAGTCTCGCGGTCAAGAGGTGAAGGTGCGATAGGTGATC-3′ E178V 425′-CAGTAAAGATAGTCTCGCGGTCAACAGGTGAAGGTGCGATAGGTGATC-3′ R180F 435′-GAAGGCTCAGTAAAGATAGTCTCAAAGTCCTCAGGTGAAGGTGCGATAG-3′ R180I 445′-GAAGGCTCAGTAAAGATAGTCTCGATGTCCTCAGGTGAAGGTGCGATAG-3′ R180L 455′-GAAGGCTCAGTAAAGATAGTCTCAAGGTCCTCAGGTGAAGGTGCGATAG-3′ R180V 465′-GAAGGCTCAGTAAAGATAGTCTCAACGTCCTCAGGTGAAGGTGCGATAG-3′ R180M 475′-GAAGGCTCAGTAAAGATAGTCTCCATGTCCTCAGGTGAAGGTGCGATAG-3′ Y276H 485′-GATATACCTCGATGTGTGAAGCGCCGTGGTTAGCAACAAGAAGGCGACCAG-3′ Y276F 495′-GATATACCTCGATGTGTGAAGCGCCAAAGTTAGCAACAAGAAGGCGACCAG-3′

TABLE 2 Primers used for PCR-based site-directed mutagenesisof Ciona intestinalis DFPase. Bold letters representsites of directed mutagenesis. SEQ ID Mutation NO. Mutagenic primerE173F 50 5′-GAAGATCGTCGTGCGGTCGAATACTGAAGGAGCAACAGGTGAG-3′ E173V 515′-GAAGATCGTCGTGCGGTCAACTACTGAAGGAGCAACAGGTGAG-3′ R175A 525′-CTCCGCGAAGATCGTCGTAGCGTCCTCTACTGAAGGAGCAACAG-3′ S60L 535′-CTCGCGGTTGTCGTCAGCAAGCTCCATAGGAGCTACTGCATAG-3′ S60R 545′-CTCGCGGTTGTCGTCAGCGCGCTCCATAGGAGCTACTGCATAG-3′

Organophosphorous Hydrolase Expression

Chloramphenicol resistant Bacillus subtilis transformants harboring theHis-tagged organophosphorous hydrolase genes described above wereinoculated into 100 ml growth medium in 250 ml Erlenmeyer flasks.Cultures were grown for 3 days at 30° C. and 250 rpm.

Example 2 Purification

Cells were harvested from the cultures by centrifugation at 5000 rpm for15 min and supernatants filtered through a 0.22 μm bottle top filter(Nalgene). Solid MES and imidazole were added to the followingconcentrations: 10 mM imidazole and 0.5 mM MES. pH was adjusted to 7.6and the solution purified using a chelating sepharose FF columnpreloaded with Cu²⁺ on a Äkta Explorer system. Elution was performedstep-wise with increasing imidazole concentrations (0%-100% of 500 mMimidazole).

Fractions belonging to the same peak were pooled, concentrated andbuffer-exchanged into 50 mM TRIS pH 7.0 using Amicon Ultra centrifugalfilter devices with a 10 kDa cut-off.

Example 3 Measurement of Organophosphorous Hydrolase ActivityOrganophosphorous Hydrolase Activity

The organophosphorous hydrolase activity of the Ciona savigny DFPase wasdetermined either by a pH stat assay as described in Blum et al., JACS,128 (2006): 12750-12757, or using in situ Fourier transform infraredspectroscopy as described in Gäb et al., Anal Biochem, 385(2009):187-193. In the pH stat assay DFP hydrolysis was determined by ameasuring the release of fluoride ions at 298 K in a nitrogenatmosphere. The assay was performed in 3 ml at pH 7.5, containing 10 mMNaCl and 10% acetonitrile. The reaction was initiated by addition of 2microliter of 0.5 mg/ml organophosphorous hydrolase. Initial velocitieswere determined at eight different substrate concentrations (0.5-10 mM),and corrected for the uncatalyzed rate of DFP hydrolysis. In situFourier transform infrared (FTIR) spectroscopy was used to measurereal-time reaction rates of the nerve agent substrates when these werehydrolyzed to the corresponding phosphoric and phosphonic acids.

Hydrolysis of dihydrocoumarine was followed at 25° C. at 235 nm in aspectrophotometer by addition of purified organophosphorous hydrolase toa solution containing 1 mM dihydrocoumarine in 50 mM Tris, 2 mM CaCl₂,pH 7.5. The specific activities of hydrolysis of dihydrocoumarine forthe organophosphorous hydrolases when calculated as decrease inabsorbance at 235 nm per minute per mg of protein was calculated to be:3 U/mg for Ciona savigny.

Activity Tests

The organophosphorous hydrolases were tested with the followingG-agents: DFP, Soman, Cyclosarin and Sarin. The Ciona savignyorganophosphorous hydrolase showed activity against all four G-agents.

TABLE 3 Specific activities. One U is defined as the hydrolysis of 1μmol substrate per minute. Specific activity of Ciona savigny Substrateorganophosphorous hydrolase in U/mg DFP (1.79%) 118 Sarin (1.97%) 101Soman (1.89%) 48 Cyclosarin (1.9%) 75 Coumarine 3The enzymatic activities of Ciona savigny and Ciona intestinalisorganophosphorous hydrolase towards Soman and VX were also performedusing NMR spectroscopy. Experiments were performed in 50 mM TRIS buffer,2 mM CaCl₂, 20% D₂O pH 7.0. The specific activities of hydrolysis ofSoman for the organophosphorous hydrolases were calculated from standardcurves for the Soman break-down product pinacolyl methylphosphonic acid(PMPA). The specific activities of hydrolysis of VX for theorganophosphorous hydrolases were calculated from standard curves forthe VX break-down product ethyl methylphosphonic acid (EMPA).

TABLE 4 Specific activity. One U is defined as the hydrolysis of 1 μmolsubstrate per minute. Specific activity of Ciona intestinalis Substrateorganophosphorous hydrolase in U/mg Soman 25.5 VX 0.012

TABLE 5 Specific activity. One U is defined as the hydrolysis of 1 μmolsubstrate per minute. Specific activity of Ciona savigny Substrateorganophosphorous hydrolase in U/mg Soman 17.8 VX 0.017VX-hydrolysis of the organophosphorous hydrolases were determined in acolorimetric assay based on the detection of free thiols with DTNB(5,5′-dithiobis-2-nitrobenzoate), as described in Broomfield et al.,CBMTS III Conference Proceedings, Spietz, Switzerland, May 7-12 (2000).

Principle of DTNB assay for detection of organophosphorous hydrolasecatalysed VX breakdown.

In the DTNB assay the organophosphorous hydrolase catalyzed VXhydrolysis is measured as the accumulation of 5-thio, 2-nitrobisbenzoate at 412 nm. The assay was performed in 200 μl at pH 7.0,containing 50 mM TRIS buffer, 2 mM CaCl₂, 0.2 mM DTNB and 3.4 mM VX and30 μg organophosphorous hydrolase enzyme (Ciona intestinalisorganophosphorous hydrolase or Ciona savigny organophosphoroushydrolase).

VX hydrolysis of all variants of CINT and CSAV DFPases were measured.The results in Tables 6 and 7 are the average of three independentreplicates.

TABLE 6 Relative VX breakdown activity of Ciona savigny DFPase variants.Measured as the accumulation of 5-thio, 2-nitro bisbenzoate at 412 nm.30 μg of CSAV DFPase enzyme was used in each assay. Relative activitycompared to CSAV Mutation SEQ ID NO. DFPase activity wildtype 2 1.0 M63A9 0.7 M63G 10 0.8 R107I 11 0.5 R107V 12 0.8 R107L 13 0.6 A109S 14 0.4A109C 15 0.7 E178F 16 0.7 E178I 17 0.8 E178L 18 0.7 E178V 19 0.8 R180F20 0.9 R180I 21 1.8 R180L 22 1.4 R180V 23 0.4 R180M 24 1.0 Y276H 25 0.6Y276F 26 0.5

TABLE 7 Relative VX breakdown activity of Ciona intestinalis DFPasevariants. Measured as the accumulation of 5-thio, 2-nitro bisbenzoate at412 nm. 30 μg of CINT DFPase enzyme was used in each assay. Relativeactivity compared to CINT Mutation SEQ ID NO. DFPase activity wildtype 41.0 E173F 27 0.4 E173V 28 0.6 R175A 29 0.9 S60L 30 1.2 S60R 31 0.4The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1. An isolated polypeptide having organophosphorous hydrolase activity,selected from the group consisting of: (a) a polypeptide having at least65%, e.g., at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to the mature polypeptide of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; (b) a polypeptide encoded by apolynucleotide that hybridizes under medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7,or (ii) the full-length complement of (i); (c) a polypeptide encoded bya polynucleotide having at least 65%, e.g., at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,or SEQ ID NO: 7; (d) a variant of the mature polypeptide of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, comprising asubstitution, deletion, and/or insertion at one or more positions; and(e) a fragment of the polypeptide of (a), (b), (c), or (d) that hasorganophosphorous hydrolase activity.
 2. The polypeptide of claim 1,having at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% or 100% sequence identity to the mature polypeptide of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO:
 8. 3. The polypeptide of claim1, which is encoded by a polynucleotide that hybridizes under mediumstringency conditions, medium-high stringency conditions, highstringency conditions, or very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, or SEQ ID NO: 7, or (ii) the full-length complement of (i). 4.The polypeptide of claim 1, which is encoded by a polynucleotide havingat least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to the mature polypeptide coding sequence of SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO:
 7. 5. Thepolypeptide of claim 1, comprising or consisting of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9 to SEQ ID NO: 26, or SEQID NO: 27 to SEQ ID NO: 31; or the mature polypeptide of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9 to SEQ ID NO: 26,or SEQ ID NO: 27 to SEQ ID NO:
 31. 6. The polypeptide of claim 1, whichis a variant of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 6, or SEQ ID NO: 8, comprising a substitution, deletion,and/or insertion at one or more positions.
 7. The polypeptide of claim1, which is a fragment of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 9 to SEQ ID NO: 26, or SEQ ID NO: 27 to SEQ ID NO:31, wherein the fragment has organophosphorous hydrolase activity.
 8. Acomposition comprising the polypeptide of claim
 1. 9. The composition ofclaim 8, wherein the composition is a micro emulsion or a lotion. 10.(canceled)
 11. A method for removing an organophosphorous compound,comprising contacting the organophosphorous compound with thepolypeptide of claim
 1. 12. An isolated polynucleotide encoding thepolypeptide of claim
 1. 13. A nucleic acid construct or expressionvector comprising the polynucleotide of claim 12 operably linked to oneor more control sequences that direct the production of the polypeptidein an expression host.
 14. A recombinant host cell comprising thepolynucleotide of claim 12 operably linked to one or more controlsequences that direct the production of the polypeptide.
 15. A method ofproducing the polypeptide of claim 1, comprising cultivating a cell,which in its wild-type form produces the polypeptide, under conditionsconducive for production of the polypeptide; and recovering thepolypeptide.
 16. A method of producing a polypeptide havingorganophosphorous hydrolase activity, comprising cultivating the hostcell of claim 14 under conditions conducive for production of thepolypeptide; and recovering the polypeptide.
 17. A transgenic plant,plant part or plant cell transformed with a polynucleotide encoding thepolypeptide of claim
 1. 18. A method of producing a polypeptide havingorganophosphorous hydrolase activity, comprising cultivating thetransgenic plant or plant cell of claim 17 under conditions conducivefor production of the polypeptide; and recovering the polypeptide.